Most prostate cancers initially respond to androgen deprivation therapy but eventually progress to castration-resistant disease. Once castration-resistant prostate cancer (CRPC) arises, it cannot be effectively treated. This fact creates a need to better understand mechanisms of CRPC to enable new therapies for patients with advanced disease. In preliminary studies, we used transposon-based mutagenesis of androgen-sensitive prostate epithelial cells to derive sub-lines that were resistant to absence of androgens or the presence of bicalutamide. Molecular analysis of transposon insertions in the castration-resistant sub-lines identified MAGI2 as a potential driver of the castration resistant phenotype. Previous whole genome sequencing of human prostate cancers also found genomic rearrangements in MAGI2. Both the transposon insertions observed in our study and the rearrangements observed in human prostate cancers could potentially cause both loss of full-length MAGI2 expression and gain of expression for a truncated N-terminal fragment of MAGI2. Data mining the results of published RNA-seq studies of CRPC also identified candidate fusion transcripts involving the MAGI2 N-terminus. These data fit with additional preliminary studies of protein expression for the N-terminus of MAGI2 on human prostate cancer tissue microarrays. In these studies we observed that high expression of the MAGI2 N-terminus was associated with an increased risk of death from prostate cancer. In light of these findings, we hypothesize that over-expression of the N-terminus of MAGI2 drives progression to CRPC. We will test this hypothesis via gain- and loss-of-function approaches in cell line and mouse models (Aim 1) and by further investigating expression of the MAGI2 protein and candidate MAGI2-regulated pathways in human prostate cancer (Aim 2). This project will have high-impact outcomes for the effort to combat prostate cancer by furthering the goal of understanding mechanisms of resistance for men with prostate cancer. This project will also further the goal of distinguishing aggressive from indolent disease because our preliminary studies support the concept that MAGI2 and MAGI2-regulated pathways as potential biomarkers to distinguish aggressive from indolent disease.